Some ideas:

Increase the Tm, you can try with 62ºc or 64ºc, if you have smear but you band is still there, increase more and more.

Anotehr reason can be excess of MgCl2 in the PCR reaction if you are using Taq

Excess of template in the reaction is common too, like you can see in your gel, with a lot of temple you have more smear, in fact in the Nº7 (from left to right) you have you band and no smear, but yes some primers dimers or inespecific.

How many cycles are you doing ??? you can try with less cycles

Experiment with adjusting 1 variable at a time to see if it helps.

Another non molecular reason could be faulty wells in the heating block of your thermalcycler. If the reactions are not cycling at the correct temperatures then something that has worked before may suddenly no longer work correctly.

Good luck !!

Hi Sun,

Your PCR may need optimization...is it possible for you to upload the gel image so that users can submit comments useful to your experimentation approach..

bye

THANK U . This is the picture of pcr result. from right to left, the concentration of pcr temple are ten fold diluted.

Some ideas:

Increase the Tm, you can try with 62ºc or 64ºc, if you have smear but you band is still there, increase more and more.

Anotehr reason can be excess of MgCl2 in the PCR reaction if you are using Taq

Excess of template in the reaction is common too, like you can see in your gel, with a lot of temple you have more smear, in fact in the Nº7 (from left to right) you have you band and no smear, but yes some primers dimers or inespecific.

How many cycles are you doing ??? you can try with less cycles

Experiment with adjusting 1 variable at a time to see if it helps.

Another non molecular reason could be faulty wells in the heating block of your thermalcycler. If the reactions are not cycling at the correct temperatures then something that has worked before may suddenly no longer work correctly.

Good luck !!

Thank u!

I have use Taq enzyme. Many of the students use Taq when they pcr, and their results are normal without smear. Should I change another enzyme?

Actually, the given Tm of the primer is 55 , I have tried 60. Increasing the Tm help to decrease primer dimers or smear?

The concentration of the right is 100fg, I added o.5ul in the pcr total volumn to 20ul. Is this too much?

Would u like to suggest me about the pcr template? If I want to have a pure template, would I use plasmid OR extracted from gel?

Hi Miao, did you try to add some of DMSO in your PCR mix? This already happened to me and DMSO solved my problem. I suggest to you to do one reaction with e without DMSO and see if this increases the quality of your PCR products on gel. Good luck!

Hi Miao

I have seen the gel and as suggested by others I also feel that you need to optimize your PCR reaction conditions and also thermal cycling conditions to optimize it.....

1) you can decreas the template further.....if your are doing a gel extraction of template for PCR...I'll suggest you to do a 'Band-Stab' using a fine needle for taking out a template...it works out most of the time.....

2) amplifiy the annealing temperature......

3) reduce the time of extension for amplification.......

4) use less amount of Taq polymerase.....

5) decrease the amount of primers

6) use some denaturant as suggested by Fernanda above

7) Try to do a hot start PCR....to reduce the non-specific amplification

follow these steps and it should be a clean amplification of your GOI

bye

The smear in the gel can be due to the following reasons;

1. Degradation of isolated RNA (avoid repeated freeze thaw cycle and minimum exposure to non sterile environment. Use RNase free reagents). Convert RNA to cDNA and store at - 20C. cDNA is more stable than RNA.

2. The elongation time is less (In your case, this could be a reason). Due to this reason, you ll be getting different lengths of DNA in the exponential phase of the amplification and after final elongation step (final elongation time = 5 to 10 min), you ll end up getting smears.

3. The annealing temperature - Run a gradient PCR with 3 temps below Tm, Tm and 2 temps above Tm as the annealing temp. Also if you are using 15 sec as your annealing cycle time, increase it up to 30 sec.

4. No of Cycles - I've read papers in which two step RT-PCR was performed with 45 cycles. But I would suggest you to perform your PCR between 30-35 cycles.

5. Genomic DNA contamination (Which is not there in your sample).

6. During extraction of the cDNA from the low melting point agarose gel, over heating could denature the DNA and DNase contamination is possible. If you are using target gene specific primer, then you can avoid this step and use the total cDNA to perform PCR.

Primer dimers will not result in a smear that you see in the first well from the right. Dimers will be seen below 100bp band in the gel. Try to change elongation time, standardize the annealing temp and no of cycles.

All the best for your experiments!

Thanks to all of u for providing me these suggestions. I'll try soon. Hope it will work. Thanks again!

Hi Miao

you can solve PCR outcome smear?

my problem is similar you

you can used AMPLIQON ready master mix for PCR reactions. If the problem is not solved , the amount of DNA cut in half. Or you could mixed 1ul cDNA with 2 ul DDW and then 1ul uptake Use as a template. Another way of putting Tm gradiant programe in thermal cycler from 52 c to 68 c with 2c difrences.

Hi Miao

I really hope your problem was resolved, it had been a long time.

Please inform us by your update ..

in addition to all comment above ,i Just have some points to explain:

You must re optimize your reaction step by step and do not change more than one parameters by one reaction.

What is your product size? i think that you get non-specific band.

Are you measure the purity and concentration of your sample before PCR?

And you said that you used the annealing of 60 why! Tm of your primer is 55 so you should to try (54-53-52-51-50) not more, Generally, an annealing temperature about 5°C below the lowest Tm of the pair of primers .so i don't prefer to use more than 54 because you know the melting temperature (Tm) is the temperature at which one-half of a particular DNA duplex will dissociate and become single strand DNA. The stability of a primer-template DNA duplex can be measured by its Tm. Primers with melting temperatures in the range of 52-58°C generally produce better results .

finally it is a good recommend to use Dnase/Rnase free water and be insure that it's not be contamination.

with my best regards

Good luck

I had a few problems in last time with a few complex templates with repetitive elements and made good experience with addition of 75 µg/ml BSA or even better 1 M betaine anhydrate (from 5 M stock in water), which strongly shifts the PCR to the specific band.

we are doing pcr analysis.  the pcr product is amplified but we cannot visualise clear bands in  superior grade agarose (1.2%). please help me out .

 thank you.

hi dear Miao Lee

I think your problem is dependent on broken DNA.

maybe, your DNA extraction was not gentle enough.

My suggestion is decreased concentration of DNA teample.

Hi

Any one please can suggest to me how can i solve this problem? a smear of genomic DNA with the band ?? size of my pcr product around 1kb, what i did here is gradient PCR the temperature from 58C to 65C

Huda Alkatib

Maybe too many cycles?

https://academic.oup.com/nar/article-abstract/19/18/5079/1078749?redirectedFrom=PDF